Thomas, Fitzgerald M.
Note: / In section A a review has been made of single crystal studies done from 1960-1973. This review has been put into tabular form (see tables I, 2, 4-9). A critical analysis and INDO calculations of some of ther adicals are reported....
14 October 2013
Computer simulations for investigating protein folding and evolution are presented. In chapter 1, an all-atom model with a knowledge-based potential is used to study the folding kinetics of Formin-Binding protein. We study the folding kinetics by performing Monte Carlo simulations. We examine the order of formation of two beta-hairpins, the folding mechanism of each individual beta-hairpin, and transition state ensemble (TSE) and compare our results with experimental data and previous computational studies. Further, a rigorous Pfold analysis is used to obtain representative samples of the TSEs showing good quantitative agreement between experimental and simulated phi values.
The three major mechanical components of cells are the biopolymers actin, microtubules, and intermediate filaments. Cellular processes are all highly reliant on the mechanics of the specific biopolymers and the networks they form, rendering necessary the study of both the kinetics and mechanics of the cytoskeletal components. Here, we study the in vitro mechanics of actin and composite actin/vimentin networks, and the effect of various actin-binding proteins on these networks. / Engineering and Applied Sciences
Allele-specific detection of single mRNA molecules in situ and the study of transcriptional regulationHansen, Clinton Hugh January 2014 (has links)
We developed a method for fluorescence in situ identification of individual mRNA molecules, allowing quantitative and accurate measurement, in single cells, of allele-specific transcripts that differ by only a few nucleotides. By using a combination of allele-specific and non-allele-specific probe libraries, we achieved >95% detection accuracy. We used this technique to investigate the allele-specific stochastic expression of Nanog, which encodes a pluripotency factor, in murine embryonic stem cells. We find that Nanog does not switch between monoallelic and biallelic expression when culture conditions are altered. We next worked towards adapting our allele-specific single molecule mRNA fluorescent in situ hybridization technique to detect early expression of the immunoglobulin kappa gene in Pre-B cells. Mature B cells only express a single allele of the immunoglobulin kappa gene, and assaying allele-specific expression in single cells will allow the study of the mechanism behind this choice. We also developed a theoretical model of cell specification in the mammalian inner ear using single-molecule mRNA expression data. During mammalian hair cell development, prosensory cells acquire a spatial pattern of distinct cellular fates. This process is dependent upon the expression of the transcription factor Atoh1, and is mediated by Notch signaling between neighboring cells. We find that both the Notch ligand and transcription factor Atoh1 are expressed in an extended region before turning off in non-hair cells. Our model reveals that this extended pattern creates a system that can suppress extraneous expression over a large region and is robust to movement of prosensory cells as the cochlea extends, especially in the case of a limited time window for specification. Our model can also explain the two types of expression patterns of Atoh1 that are observed.
Single-molecule techniques to probe the dynamic gene regulatory network formed by core pluripotency circuit in embryonic stem cellsLin, Ya January 2014 (has links)
This work investigates the dynamics of gene regulatory network formed by Oct4, Sox2 and Nanog in embryonic stem cells (ESCs). Despite a large number of existing studies on stem cells, current technologies used often force a compromise between quantification of gene expression via bulk measurements and qualitative imaging of cell heterogeneity. There are few options that allow for accurate and quantitative single-cell analysis that is robust yet not associated with a high degree of technical difficulty or obscured by amplification. Here, we adapted a high resolution, single-molecule RNA fluorescent in situ hybridization technique (smFISH) to study gene expression of the core pluripotency circuit upon various types of perturbations such as differentiation, induction or knockdown of one of the three pluripotent factors. We used previously-published smFISH procedures as our initial template for investigating gene regulatory dynamics of the core pluripotency circuit during those perturbation assays. To obtain a more comprehensive picture of the regulatory circuit, we developed a modified smFISH strategy to measure mRNA and protein expression simultaneously in single ESCs. By incorporating a novel modification into the smFISH technique which allows accurate quantification of transcripts that differ by short sequences, we managed to identify a few interesting features of the core pluripotency circuit. Taken together, we demonstrated our ability to perform single-cell, single-molecule assays that reveal highly quantitative information in unprecedented detail.
Van Benschoten, Andrew Holland
31 July 2015
<p> Understanding the physical basis of enzyme dynamics is a major challenge in biology. Although modeling the motion of individual atoms is straightforward, combining these movements into descriptions of macromolecular function proves more difficult. X-ray crystallography produces atomic-level visualizations of an ensemble of countless molecules; however, current methods capture only the average protein conformation and thus cannot completely describe the underlying dynamics. A parallel source of information, diffuse scattering, is present in diffraction images and directly reports on correlated atomic motions. </p><p> I created experimental and computational tools to measure macromolecular diffuse scattering and compare it against hypotheses of correlated motion. The first tool, <i>phenix.diffuse</i>, calculates diffuse scattering patterns from known structural ensembles. I applied this software to the refinement technique <i>Translation-Libration-Screw</i> and solved a pre-existing degeneracy within the predicted motion of glycerophosphodiesterase GpdQ. Surprisingly, I also uncovered a fundamental flaw in the implementation of TLS refinement in structural biology software, revealing unphysical motions to be present in nearly 25% of all known macromolecular structures. </p><p> Next, I developed the comprehensive pipeline <i>DIALS-LUNUS</i> for the measurement of macromolecular diffuse scattering. This system was applied to crystals of the proline isomerase cyclophilin A (CypA) and trypsin, ultimately producing high-resolution diffuse maps of both proteins. These maps were compared to several distinct models of motion that were previously indistinguishable to crystallographic techniques. By comparing the experimental data to each predicted diffuse scattering pattern, I was able to successfully identify the most probable mechanism of motion. Ultimately, these studies provide a new avenue of exploration in the pursuit of understanding molecules as dynamic entities.</p>
Structure, nucleocapsid affinity, and mechanistic implication of the 1 x 3 internal loop in SL1 from the 5'-leader of HIV-1 RNAYuan, Yiqiong Borer, Philip N. January 2004 (has links)
Thesis (PH.D.) -- Syracuse University, 2004. / "Publication number AAT 3132723."
Thesis (PH.D.) -- Syracuse University, 2006 / "Publication number AAT 3241852."
Liu, Julia Chang
04 June 2016
This work comprises analyses of cell fate decision-making in response to DNA damage. DNA damage is a ubiquitous threat to genomic stability, and depending on the type and extent of the damage, can lead to widespread changes in cell function as well as cell death. How apoptosis, or programmed cell death, is triggered in damaged cells was studied in different cell types for different types of damage.
Robinson, Andrew James
01 January 1981
The administration of glucocorticoids has been shown to induce atrophy and weakness in whole skeletal muscle. These effects frequently appear to be more pronounced in pale muscle than in red muscle. The present study was undertaken to examine the effects of steroid treatment on the contractile, electrical, and fatigue properties of single meter units in pale and red cat hindlimb muscles. Single motor units in the medial gastrocnemius (MG) and soleus muscles were isolated using techniques developed by McPhedran, et al., (1965). The properties of a large number of motor units were examined in normal animals and were compared to those of single units from the muscles of steroid-treated (3-4 mg. of triamcinolone acetonide per kg. per day for 10 -16 days) animals. Motor units examined in both control and experimental animals were classified by type according to criteria developed by Burke, et al., (1973,1974). The results show that steroids produce alterations in the strength- and speed-related properties of motor units which are more pronounced in fast-twitch than in slow-twitch units. The mean maximum tetanic tension (Pmax) for types FF (fast-twitch, readily fatiguable) and FR (fast-twitch, fatigue-resistant) units in MG were reduced by 63% and 71% respectively; The Pmax for type S units in MG was reduced by 25% as a result of steroid treatment but was unchanged for type S units in soleus. The mean maximum rate of rise of tetanic tension (dP/dt) for the three classes of motor units in MG was reduced in a pattern similar to that for tetanic tension. The dP/dt for types FF, FR, and S units in MG were reduced by 79%, 54%, and 25% respectively. The dP/dt for soleus units was increased by 73% as compared to control soleus units. Steroid treatment lengthened the mean twitch contraction time (CF) of FF units and shortened the CT for type S units in both MG and soleus. The integrated EMG signals elicited from FF and FR units were unchanged following steroid treatment but were significantly increased for type S units in both pale and red muscles. Steroid administration did not markedly alter the susceptibility to fatigue of the three classes of units in MG or in soleus units. There were however fewer MG units with intermediate fatigue indeces in steroid-treated animals. These finding suggest that those units most frequently activated in muscular contractions are the least susceptible to steroid-induced changes in their contractile properties and vice versa. Since the overall frequency of activation of motor uﬁits is determined by cell size, these results imply that the Size Principle (Hennemann, et al., 1965) can be extended to not only explain the recruitment order of motor units in muscular contractions but also to account for the patterns of motor unit involvement in steroid-induced myopathy.
Page generated in 0.1034 seconds